DISEASES OF AQUATIC ORGANISMS Vol. 38: 33-38, 1999 Published October 11 Dis Aquat Org P P Epizoic amoebae from the gills of turbot Scophthalmus maximus Iva ~ykova'~:Antonio ~igueras~,Beatriz Novoa2 'Institute of Parasitology, Academy of Sciences of the Czech Republic. BraniSovska 31,37005 ceske Bud6jovice. Czech Republic 'Concejo Superior de Investigaciones Cientificas, Instituto de Investigaciones Marinas, Eduardo Cabello 6,36208 Vigo, Spain ABSTRACT: Species of amoebae belonging to the genera Platyamoeba Page, 1969, Vannella Bovee, 1965 and Flabellula Schaeffer, 1926 were found to accompany Paramoeba sp., the agent of amoebic gill disease (AGD),in cl~nicallydiseased turbots. The same community of epizoic gymnamoebae was found on the gills of turbots which revealed no gill abnormalities but slight behavioral signs indicat~veof sub- optimal health status. The assemblage of the above-mentioned free-living amoebae capable of colo- nizing gill tissue of turbots was supplemented with species recognized in samples fixed from primary isolates for transmission electron microscopy. The pathogenic potential of epizoic gill amoebae in turbots is discussed. KEY WORDS: Epizoic amoebae . Gill infection Scophthalmus maximus. Mariculture INTRODUCTION object of the first screening was the group of clinically diseased turbots as described In the paper presenting Amoebic gill disease (AGD) of turbot Scophthalmus the agent of AGD (Dykova et a1 1998a). The second maximus (L.) was diagnosed for the first time in 1995 screening covered a group of the same hosts with clin- (Dykova et al. 1995). Due to difficult cultivation, ically healthy gills. In the first screening, 14 turbots generic assignment of the agent was published later from the stock with AGD were sampled; 4 of these had (Dykova et al. 1998a), along with a note on other clinical symptoms of AGD. The second screening was strains of free-living amoebae which accompanied carried out in 2 farms and included a total of 72 turbots: Paramoeba sp. In the same year Leiro et al. (1998) pre- the gills of 24 turbots of the size category 12 to 20 cm sented a study of Platyamoeba sp., which they claimed were sampled in farm No. 1, which experienced AGD caused AGD in farmed turbots from the same region as in 1994 (Dykova et al. 1995), and 48 turbots were reported in previous papers of Dykova et al. (1995, sampled in farm No. 2, where mortalities caused by 1998a). Unfortunately, histopathology of AGD was not AGD occurred in 1996. This group of turbots of the size studied in detail by Leiro et al. (1998), and the alleged category 12 to 15 cm comprised 20 specin~enswith agent of AGD was shown in situ in a single semithin optimal health status and 28 with slight behavioral section of a small sample of gill tissue. signs indicative of suboptimal health condition. No gill The aim of this communication is to disclose the abnormalities were detected in this group of fish. diversity of amoeboid organisms capable of colonizing Amoebae isolated from samples of turbot gill tissue the gills of turbots. were characterized and compared. In total, 53 strains were examined with the light microscope, and 27 of them were also examined with the electron micro- MATERIALS AND METHODS scope. The methods of isolation, culture and cloning as well as fixation for transmission electron microscopy The data presented are based on 2 successive (TEM) were the same as described in previous papers screenings in turbot sea farms in 1996 and 1998. The (Dykova et al. 1995, 1998b). Some of the cultures in liquid medium as well as on agar plates were rnain- tained for several weeks only, after which they per- 8 Inter-Research 1999 Resale of full article not permitted 34 Dis Aquat Org 38: 33-38, 1999 ished due to bacterial overgrowth. Non nutrient agar range of greatest dimension between 22.0 and 45.0 pm (Bacto agar, Difco) used for subculturing was seeded (such as, e.g. P. bursela Page, 1974; P. mainensis Page, with autoclaved Pseudomonas sp. 1971; P. flabellata Page, 1974; P. australis Page, 1983; and P. calycinucleolus Page, 1974). All the mentioned species were described from localities distant from the RESULTS area of our studies. Similarly, Page (1983) stressed a great intraspecific variation in Platyamoeba at light In addition to Paramoeba sp., the agent of AGD, 20 and electron microscopical levels. This, along with our strains of other amoebae were isolated from the gill own experience, confines our assignment of the clonal tissues of 7 out of 14 turbots sampled during the first strains to the generic level only until molecular analy- screening in 1996 in a turbot farm in Northwest Spain. sis is done. This idea was supported by TEM investiga- These 7 positive turbots included 4 with clinically tion which revealed slight differences in the thickness manifested AGD. of the glycocalyx of individual strains (Figs. 17 to 19). Twenty amoeba strains were isolated from the gills Culture conditions were identi.cal and the differences of 17 out of 24 turbots examined in 1998 from farm in the thickness of the glycocalyces could be attributed No. 1. Thirteen strains, including 5 Paramoeba strains, to different methods of fixation rather than real differ- were isolated from the gills of 8 out of 28 examined tur- ences in ultrastructure of individual clones. bots, which in farm No. 2 revealed no gill changes and Strains belonging to the genus Vannella Bovee, 1965 only slight behavioral alterations. Isolation attempts in were less numerous: 4 out of 22 strains of flattened the group of 20 turbots with certified optimal health amoebae analysed with TEM were assigned to this status from farm No. 2 gave negative results. genus. All of them accompanied the agent of AGD. Among 53 strains isolated from the gills of 32 out of Although the greatest dimensions of trophozoites and 86 examined turbots, the most numerous (48 strains) the type of glycocalyx (Figs. 12, 13 & 16) complied with were flattened (discoid or fan shaped.) amoebae. They the size range of V. caledonica Page, 1979, V. septen- could easily be distinguished from Paramoeba by their tn'onalis Page, 1980 and V anglica Page, 1980, we also shape and a well-developed hyaloplasmic region in restricted the diagnosis to the generic level until non- trophozoites (Figs. 1 to 15). While on agar plates, morphological criteria can be applied. trophozoites of individual strains revealed polymor- Two out of 22 analysed clonal strains were assigned phism, especially in the zones of intensive multiplica- to the genus Flabellula Schaeffer, 1926 (Figs. 14 & 15). tion; the shape of trophozoites observed in hanging- The greatest dimensions of the trophozoites fitted in drop preparations was more or less uniform. Shape the range given by Page (1983) for trophozoites of F. divided strains of flattened amoebae into 2 groups. The citata Schaeffer, 1926 or F calkinsi (Hogue, 1914). first group included strains with trophozoites possess- Five Paramoeba strains were isolated from the gills ing a veil-like extensive hyaloplasmic region (Figs. 1 to of 5 turbots with slight behavioral abnormalities. Three 13) with a smooth rim. The trophozoites of the second strains were successfully cloned and subcultured. When group were characterized by an irregular outline of observed on the surface of agar plates and in hanging- hyaloplasm (Figs. 14 & 15). drop preparations, trophozoites were rather flattened, Taxonomic analysis based on light microscopy and with a narrow hyaline zone and digitiform pseudo- TEM of 22 clonal strains representative of small, pods. One or 2 parasomes were observed in the close medium, large and giant size categories of flattened vicinity of the nucleus. The active locomotive forms trophozoites revealed the presence of species belong- were longer than broad, having a minimum length of ing to 3 genera. Sixteen out of 22 analysed strains of 14 to 15 pm and a maximum length of 27 to 33 pm flattened amoebae were assigned to the genus (Figs. 20 to 22). The average breadth was 6.0 to 8.7 pm. Platyamoeba Page, 3.969. Ten out of 16 Platyamoeba Floating forms had a rounded central mass and long strains were assigned to the group of small and radiate pseudopods. No cysts were formed by any of medium sized species of the genus which includes, these strains. The basic diagnostic features as well as e.g. P. longae Sawyer, 1975, P. murchelanoi Sawyer, details of ultrastructure observed in 3 Paramoeba 1975, P. weinsteini Sawyer, 1975, P. douversi Sawyer, strains (Fig. 23) were identical with P pemaquidensis 1975, P leei (Sawyer, 1975) Page, 1983 and P. plun'nu- Page, 1970. When compared with trophozoites of 2 cleolus Page, 1974 listed in Page (1980, 1983). The strains of P. pemaquidensis isolated from the Mediter- greatest dimensions of trophozoites of these clonal ranean area and cultured under the same conditions, a strains (Figs. 1 to 7) did not exceed the range 11.4 to difference in size of trophozoites but not in ultrastruc- 21.5 pm Trophozoite size allowed 6 out of 16 analysed ture was noticed. The average length and breadth of Platyamoeba strains to be categorized in the group of the 3 Paramoeba strains under study were smaller than large and giant species (Figs. 8 to 11) which have a those of the Mediterranean strains, but larger than Dykova et a1 : Epizoic amoebae from turbot gills 35 I. F?' 'm7y;,A., .+ "C' -R.. ... , .." J:r --;L*,*,; 2 d.5#-. 4 $9:. I' '#: $:..-.. ,& ,; ":v. ..:.- - p!;*\ - .- -% ., I 'Y I K-;&.;pp/ .;,c, Figs. 1 to 19. Trophozoites of flattened amoebae isolated from gills of turbots. Figs. 1 to 7. Trophozoites of Platyanloeba spp., rep- resentat~vesof the group of small and med~umsized species of this genus, as seen in hanging-drop preparations.